Summary: Direct cytotoxic effects associated with cell-free hemoglobins (Hb) developed as oxygen therapeutics have been ascribed to redox reactions between Hb and NO. We have investigated the impact of diaspirin cross-linked Hb (DBBF-Hb), a blood substitute, on cell signaling pathways events that are modulated by NO and other biological peroxides (i.e. H2O2, LOOH, and ONOO-) in vitro. Bovine aortic endothelial cells (BAECs) subjected to severe hypoxia expressed hypoxia-inducible factor (HIF-1a) in a time course that paralleled the expressions of phosphotyrosine (pY) and heme oxygenase (HO-1). Co-incubation of the oxy form (HbFe2+) with hypoxic BAECs resulted in an increase in the expression of HIF-1a in a manner that corresponded linearly with the loss of HbFe2+ and accumulation of the ferric form (HbFe3+). Inclusion of HbFe3+ with hypoxic BAECs produced twice as much expression in the HIF-1a and HO-1 proteins as apposed to HbFe2+ alone, and HbFe2+ plus hypoxia. In addition, higher and more persistent levels of the ferryl form (HbFe4+) (due to the consumption of endogenous peroxides) were found in the hypoxic media containing Hb. Nitric oxide (NO) released from an NO donor reduced the levels of HIF-1a in the hypoxic cells treated with either HbFe2+ or HbFe3+, but had little or no effect on the levels of HO-1 and pY proteins. We have concluded that DBBF-Hb modulates key cell-signaling pathways as it converts oxygen signal to a transduction signal that activates HIF-1a, which may result in alterations of important physiological mediators. This work has been recently submitted for publication. In human trials with current generation blood substitutes, gastrointestinal discomfort has been universally reported. The symptoms include nausea, vomiting, diarrhea, dysphagia or generalized abdominal pain. These symptoms are believed to be related to NO scavenging by Hb, causing localized spasm throughout gastrointestinal tract. We have recently shown in collaboration with Dr. Baldwin, University of Arizona that bolus injection of some chemically modified hemoglobins can increase venular permeability and mast cell degranulation in the rat mesentery. It was speculated that these hemoglobins extarvasate from the intestinal mucosal capillaries and that DBBF-Hb, in particular causes ultastructural changes in the intestinal epithelium. These changes were found to be correlated strongly with the redox chemistry of each hemoglobin. Interestingly cyanomet DBBF-Hb, in which the iron center is unable to bind NO, and other oxidants, significantly reduced leak numbers and mast cell degranulation (Am. J. Physiol. (in press))